Particles comprising decellularized omentum

ABSTRACT

A spherical particle comprising decellularized omentum being between 1 nM-300 μM in diameter is disclosed. In some embodiments, the particle comprises biological cells. In other embodiments, the particle comprises a biomolecule. Uses of the particles are also disclosed.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to particlescomprising decellularized omentum. The particles may be used for celland/or biomolecule delivery.

The omentum is a double sheet of peritoneum that extends from thegreater curvature of the stomach overlying most abdominal organs. Thistissue is highly vascularized and its fibrillar ECM is rich withcollagens, adhesive proteins and GAGs. Since GAGs bind a variety ofprotein ligands, they can serve as growth factor depots and regulate awide variety of biological activities, including developmentalprocesses, angiogenesis, and cardioprotection. Due to its uniquecomposition, the omentum also serves as a depot for adult stem cellswith regenerative potential. These stem cells are based in the omentummatrix and upon signals migrate to heal injured organs. The overallregenerative capacity of the omentum, its ability to maintain progenitorcell viability, absorb large amounts of edema fluids and limit theformation of scar tissue at the site of injury, has long beendemonstrated.

Dvir, T., et al. (Proc Natl Acad Sci USA 106, 14990-14995 (2009))teaches the utilization of the omentum to induce cell migration andblood vessel network formation in an implanted synthetic scaffold. Thesevascularized scaffolds were then re-implanted on the infarcted heart andcompletely attenuated its deterioration.

International Patent Application No. WO2009/085547 teaches thegeneration of decellularized omentum scaffolds for tissue engineering.

U.S. Patent Publication No. 20050013870 teaches a scaffold comprisingdecellularized extracellular matrix of a number of body tissuesincluding omentum. The body tissues have been conditioned to produce abiological material such as a growth factor.

U.S. Patent Publication No. 20090163990 teaches methods ofdecellularizing omentum.

U.S. Patent Publication No. 20150202348 teaches decellularized omentumfor tissue engineering.

Porzionato et al. (Italian Journal of Anatomy and Embryology, Volume116, 2011 and Eur J Histochem. 2013 Jan. 24; 57(1):e4. doi:10.4081/ejh.2013.e4) teaches decellularized omentum.

Soluble forms of decellularized extracellular matrix are known in theart as described in Acta Biomaterialia, Volume 9, Issue 8, August 2013,Pages 7865-7873 and Singelyn et al., J Am Coll Cardiol. Feb. 21, 2012;59(8): 751-763.

Additional background art includes WO2014/037942, Gilbert et al.,Biomaterials 27 (2006) 3675-3683 and Flynn et al., Biomaterials 31(2010), 4715-4724.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a spherical particle comprising decellularized omentumbeing between 1 nm-300 μM in diameter.

According to an aspect of some embodiments of the present inventionthere is provided a composition comprising:

(i) a plurality of the particles described herein; and

(ii) biological cells and/or at least one biomolecule.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating particles comprisingdecellularized omentum comprising:

(a) dispersing a composition comprising solubilized decellularizedomentum in an oil under conditions that allow generation of emulsified,decellularized omentum; and

(b) heating the emulsified, decellularized omentum to generate solidparticles of decellularized omentum, thereby generating particlescomprising decellularized omentum.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a disease or medical conditionwhich would benefit from cell transplantation in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a plurality of the particles described herein,thereby treating the medical condition.

According to some embodiments of the present invention, the particle isa microparticle.

According to some embodiments of the present invention, the particle isbetween 50-300 μm in diameter.

According to some embodiments of the present invention, the particle isa nanoparticle.

According to some embodiments of the present invention, more than 20% ofthe particle is composed of decellularized omentum.

According to some embodiments of the present invention, the omentumcomprises human omentum.

According to some embodiments of the present invention, the particleencapsulates at least one biological cell.

According to some embodiments of the present invention, the biologicalcell is selected from the group consisting of a cardiac cell, a neuronalcell, a pancreatic cell, a stem cell, a liver cell, a muscle cell, ablood cell and an immune cell.

According to some embodiments of the present invention, the particleencapsulates at least one biomolecule.

According to some embodiments of the present invention, the particlefurther encapsulates at least one biomolecule.

According to some embodiments of the present invention, the biomoleculeis selected from the group consisting of bone morphogenetic protein-2(BMP-2), bone morphogenetic protein-7 (BMP-7), transforming growthfactor beta (TGF-β), interleukin 10 (IL10), vascular endothelial growthfactor (VEGF), Insulin-like growth factor (IGF-1), stromal cell derivedfactor-1 (SDF-1), platelet derived growth factor (PDGF), neurotrophin(NT-3), dexamethasone, noradrenaline, keratinocyte growth factor (KGF),angioprotein (Ang-1), fibroblast growth factor (FGF-2) and nerve growthfactor (NGF).

According to some embodiments of the present invention, each of theparticles of the plurality of particles are of substantially the samesize.

According to some embodiments of the present invention, the particlesencapsulate the biological cells and/or at least one biomolecule.

According to some embodiments of the present invention, the biologicalcells are selected from the group consisting of cardiac cells, neuronalcells, pancreatic cells, stem cells, liver cells, and muscle cells.

According to some embodiments of the present invention, the at least onebiomolecule is selected from the group consisting of bone morphogeneticprotein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), transforminggrowth factor beta (TGF-β), interleukin 10 (IL10), vascular endothelialgrowth factor (VEGF), Insulin-like growth factor (IGF-1), stromal cellderived factor-1 (SDF-1), platelet derived growth factor (PDGF),neurotrophin (NT-3), dexamethasone, noradrenaline, keratinocyte growthfactor (KGF), angioprotein (Ang-1), fibroblast growth factor (FGF-2) andnerve growth factor (NGF).

According to some embodiments of the present invention, the methodfurther comprises separating the solid particles comprisingdecellularized omentum from the oil following the heating.

According to some embodiments of the present invention, the heating iseffected at about 37° C.

According to some embodiments of the present invention, the oil is afluorinated oil.

According to some embodiments of the present invention, the fluorinatedoil is selected from the group consisting of FC-40, FC-770, FC-70,FC-77, FC-72, FC-43, FC-3283, FC-3284, perfluoro-hexane (PFH),perfluoro-cyclohexane (PFC), perfluoro-decaline (PFD),perfluoro-perhydrophenanthrene (PFPH) and Novec/hydrofluoroether(HFE)-7500/7100/7200/71DA/71DE/71IPA/72DA/72DE.

According to some embodiments of the present invention, the oil is ahydrocarbon oil.

According to some embodiments of the present invention, the hydrocarbonoil is selected from the group consisting of light mineral oil, heavymineral oil, hexadecane, tetradecane, octadecane, dodecane, Isopar™Isoparaffinic fluids and vegetable oil.

According to some embodiments of the present invention, the compositionfurther comprises a surfactant.

According to some embodiments of the present invention, the surfactantis selected from the group consisting of pico-surf™ 1, pico-surf™ 2,perfluoro (PF)-octanol, PF-decanol, PF-tetradecanoic (TD) acid, PF-TDOEG, perfluoropolyether (PFPE)-COOH, PFPE-COONH4, PFPE-PEG, PFPE-DMP,perfluoro-short chains.

According to some embodiments of the present invention, the separatingthe solid particles of decellularized omentum from the oil is effectedby contacting the solid particles with a hydrophilic solution.

According to some embodiments of the present invention, the separatingis effected using a microfluidics device.

According to some embodiments of the present invention, the separatingis effected using a 3D printer.

According to some embodiments of the present invention, the methodfurther comprises sonicating the solid particles following theseparating.

According to some embodiments of the present invention, the compositionfurther comprises at least one biological cell.

According to some embodiments of the present invention, the at least onebiological cell is selected from the group consisting of a cardiac cell,a neuronal cell, a pancreatic cell, a stem cell, a liver cell, a musclecell.

According to some embodiments of the present invention, the compositionfurther comprises a biomolecule.

According to some embodiments of the present invention, the biomoleculeis a growth factor.

According to some embodiments of the present invention, the biomoleculeis a neuropeptide.

According to some embodiments of the present invention, the biomoleculeis a neurotransmitter.

According to some embodiments of the present invention, the omentumcomprises human omentum.

According to some embodiments of the present invention, the methodfurther comprises crosslinking the particles following the separating.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a scheme of an exemplary microfluidic device. The omentum-gelstream is dispersed by the continuous phase, the oil stream, to produceomentum-gel droplets. According to some embodiments, all microfluidicschannels are 25 μm wide and high. According to other embodiments, themicrofluidics channels are 50 μm in width and height, except for thenozzle (at the zone where the droplets are produced) are 40 μm in width.

FIGS. 2A-B are bright field (BF) images of the device dropletsgeneration zone (FIG. 2A) and the device outlet region of droplets (FIG.2B). Cells can be observed within the droplets. Bar-100 μm.

FIG. 3 is a graph illustrating the rheological properties ofomentum-hydrogel. Representative curves of storage (G′, consecutiveline) and the loss modulus (G″, dashed line) during gelation at 37° C.

FIGS. 4A-C illustrate viability (FIG. 4A) and size distribution ofencapsulated 3T3 fibroblasts cells using 5% (FIG. 4B) and 2% (FIG. 4C)pico-surf™2 surfactant. Continuous phase velocity—80 μl hr ⁻¹, dispersedphase velocity—40 μl hr ⁻¹.

FIGS. 5A-E are graphs illustrating cell viability (FIG. 5A), dropletmean diameter (FIG. 5B) and droplet size distribution of encapsulated3T3 fibroblasts cells using 20 (FIG. 5C), 40 (FIG. 5D) and 60 (FIG. 5E)μl hr ⁻¹ dispersed phase velocity. Continuous phase velocity—80 μl hr⁻¹.

FIGS. 6A-B are photographs of encapsulated cells. (FIG. 6A) BF image of3T3 fibroblasts cells immediately after encapsulation (dispersed phasevelocity—20 μl hr ⁻¹, bar-100 μm) and (B) Fluorescence image of live(green) and dead (red) cells in aqueous medium (dispersed phase velocity60 μl hr ⁻¹). Bar-100 μm, bar of enlarged images—50 μm. Continuous phasevelocity in (A) and (B) 80 μl hr ⁻¹.

FIGS. 7A-E are graphs illustrating cell viability (FIG. 7A), dropletmean diameter (FIG. 7B) droplet size distribution of encapsulated 3T3fibroblasts cells using 60 (FIG. 7C) 80 (FIG. 7D) and 100 (FIG. 7E) μlhr ⁻¹ continuous phase velocity. Dispersed phase velocity—40 μl hr ⁻¹.

FIGS. 8A-F are graphs illustrating cell viability (FIG. 8A), cell numberper droplet (FIG. 8B)) droplet size distribution (FIGS. 8C-D) and brightfield image (FIGS. 8E-F) of encapsulated 3T3 fibroblasts cells using25×10⁶ mL⁻¹ (FIGS. 8C,E) and 50×10⁶ mL⁻¹ (FIGS. 8D,F). Scale bar (FIGS.8E-F)—100 μm. Dispersed phase velocity—40 μl hr ⁻¹. Continuous phasevelocity—80 μl hr ⁻¹.

FIGS. 9A-C illustrate scanning electron microscopy of omentum-hydrogeldroplets with (FIGS. 9A-B) and without (FIG. 9C) encapsulated 3T3fibroblasts cells.

FIGS. 10A-C are immunofluorescence images of encapsulated spinal cordneurons (collagen stained green, nuclei stayed blue and β-tubulinstained red). Scale bar—100 μm.

FIGS. 11A-D are immunofluorescence images of encapsulated neonatal CMs(collagen stained green, nuclei stayed blue and α-actinin stained red),at day 8 (FIGS. 11A-B) and day 13 (FIGS. 11C-D) after encapsulation.Scale bar (A)—100 μm.

FIGS. 12A-B: Encapsulation of 3T3 fibroblasts in omentum-gel dropletsusing emulsion mixing. A. Bright field image of a droplet. B. Cellnuclei is visualized by fluorescence image of hoechst staining. 1%omentum-gel, containing 50,000 fibroblasts/ml, was dispersed in vacuumoil, 0.2% span 80.

FIGS. 13A-B illustrate encapsulation of 3T3 fibroblasts in omentum-geldroplets generated using microfluidics. A. Bright field image of thedroplets B. Droplets are visualized by fluorescence microscopy (greenautofluorescence). 0.67% omentum-gel, containing 1,000,000fibroblasts/ml, was dispersed in fluorinated-40 (FC-40) oil, 2% span 80.

FIG. 14 illustrates 1% omentum-gel droplet production using 3D inkjetbio-printing. Droplets were very homogenous in size exhibitingapproximately 40 μm diameter. The droplets were printed directly intoheavy mineral oil.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to particlescomprising decellularized omentum. The particles may be used for celland/or biomolecule delivery.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Encapsulating cells in hydrogels has shown promising results forreducing the immune response, but many preclinical and clinical trialsresults have been inconsistent because of the limited ability ofhydrogels to support cellular viability and function over an extendedtime period. Therefore, few successful products have been fullycommercialized based on these cell-encapsulation technologies.

The present inventors have now generated micro and nanoparticles made ofautologous biomaterial originating from the omentum. The omentum washarvested from subjects via a simple laparoscopic procedure. Followingdecellularization using physical, chemical or/and enzymatic processing,the extracellular matrix (ECM) of the omentum was converted into aliquid substance that was emulsified to generate omentum-ECM particlesthat were thermoresponsive and gel upon incubation at 37° C. The presentinventors have shown that cells and/or biomolecules (e.g. cytokines,growth factors, drugs etc.) and/or other nanoparticles may be entrappedin these particles (FIGS. 10A-C, 11A-D, 12A-B and 13A-B). Followinggelation, the resulting particles may serve as vehicles for protectionand release of the cargo for various applications such as tissueengineering and regeneration procedures. As the microparticles have amuch greater surface area than the same amount of gel not formulatedinto droplets, their release can be much more precisely controlled andtailored to the specific desired usage. In addition, mass transfer ofoxygen and nutrients is more efficient leading to improved cellviability.

According to a first aspect of the present invention, there is provideda method of generating particles comprising decellularized omentumcomprising:

(a) dispersing a composition comprising solubilized decellularizedomentum in an oil under conditions that allow generation of emulsified,decellularized omentum; and

(b) heating the emulsified, decellularized omentum to generate solidparticles of decellularized omentum, thereby generating particlescomprising decellularized omentum.

As used herein the phrase “decellularized omentum” refers to theextracellular matrix which supports omentum tissue organization whichhas undergone a decellularization process (i.e., a removal of all cellsfrom the tissue) and is thus devoid of cellular components.

The decellularized omentum comprises extracellular matrix (ECM)components.

The phrase “extracellular matrix (ECM)” as used herein, refers to acomplex network of materials produced and secreted by the cells of thetissue into the surrounding extracellular space and/or medium and whichtypically together with the cells of the tissue impart the tissue itsmechanical and structural properties. Generally, the ECM includesfibrous elements (particularly collagen, elastin, and/or reticulin),cell adhesion polypeptides (e.g., fibronectin, laminin and/or adhesiveglycoproteins), and space-filling molecules [usually glycosaminoglycans(GAG), proteoglycans].

Omentum may be harvested from mammalian species, such as human, swine,bovine, goat and the like. Following tissue harvesting, the tissue canbe either placed in 0.9% saline for immediate processing or stored forlater use, preferably at a temperature of about −20° C. to about 80° C.

According to a preferred embodiment, the omentum is derived from ahuman.

Methods of decellularizing omentum may be found in U.S. Patent No.20150202348 and WO2014/037942, the contents of which are incorporatedherein by reference.

According to one embodiment of the present invention, thedecellularization is carried out by:

(a) exposing the omentum to a hypotonic solution;

(b) dehydrating the omentum following step (a);

(c) extracting fat from the dehydrated omentum using polar and non-polarextraction agents following step (b);

(d) rehydrating the dehydrated omentum following step (c); and

(e) extracting cells from the rehydrated omentum following step (d).

Omentum may be harvested from mammalian species, such as human, swine,bovine, goat and the like. Following tissue harvesting, the tissue canbe either placed in 0.9% saline for immediate processing or stored forlater use, preferably at a temperature of about −20° C. to about 80° C.

According to a preferred embodiment, the omentum is derived from ahuman.

A hypotonic solution is one in which the concentration of electrolyte isbelow that in cells. In this situation osmotic pressure leads to themigration of water into the cells, in an attempt to equalize theelectrolyte concentration inside and outside the cell walls.

Preferably, the hypotonic buffer used by the method according to thisaspect of the present invention is 10 mM Tris solution at a pH of about8.0 and includes approximately 0.1% (w/v) EDTA (5 mM EDTA).

The hypotonic buffer may comprise additional agents such as serineprotease inhibitors (e.g. phenylmethanesulfonylfluoride orphenylmethylsulfonyl fluoride, PMSF) and/or anionic detergents such assodium dodecyl sulphate (SDS).

According to this aspect of the present invention, the tissue issubjected to the hypotonic buffer for a time period leading to thebiological effect, i.e., cell swelling and rupture.

Following hypotonic shock, the tissue may optionally be subjected tocycles of freeze-thawing.

The freeze/thaw process preferably comprises freezing the tissue at, forexample between −10 to −80° C., and typically at −80° C. for between2-24 hours and subsequently defrosting the tissue for about 2, 3 or 4hours until it reaches room temperature or above (for example at 37°C.). This process is carried out at least once and preferably twice orthree times in the presence of a hypotonic buffer.

Dehydration involves treating the omentum with one or more dehydrationsolvents, such one or more treatments of the omentum with a dehydrationsolvent(s) and/or such solvent(s) in solution with water. The one ormore treatments may be sequential steps in the method performed withsolutions having different ratios of dehydration solvent(s) to water,such as having gradually reduced amounts of water in the solution foreach successive treatment and the final treatment may involve the use ofpure solvent, i.e., solvent not in solution with water.

Low molecular weight organic solvents may be used for the dehydrationsolvent. In an embodiment, the dehydration solvent is one or morealcohols, such as those selected from the group consisting of methanol,ethanol, isopropanol, propanol and combinations thereof.

According to a particular embodiment, the omentum is dehydrated byrinsing once with 70% ethanol (for example for 10-60 minutes) and two tothree times in 100% ethanol for 10-60 minutes each.

After dehydration, the fat may be extracted from the omentum using atleast one polar solvent and one non-polar solvent, which may occur inone or more extraction steps.

Examples of non-polar solvents are non-polar organic solvents such ashexane, xylene, benzene, toluene, ethyl acetate and combinationsthereof. Polar solvents useful for the extraction solvent includeacetone, dioxane, acetonithle and combinations thereof. In anembodiment, the extraction solvent is selected from acetone, hexane,xylene and combinations thereof. Nonpolar solvents, include for examplehexane, xylene and combinations thereof.

Fat extraction may be conducted in fat extraction steps by contactingthe dehydrated omentum with the extraction solvents for varying periodsof time.

Preferably, the polar lipids of the tissue are extracted by washing inthe polar extraction agent (e.g. 100% acetone) between 10 minutes to 60minutes. This may be repeated a number of times (e.g. three times).Then, the nonpolar lipids may be extracted by incubating in a mixture ofnonpolar:polar agents (e.g. 60/40 (v/v) hexane:acetone solution (with 3changes) or 60/40 (v/v) hexane:isopropanol solution (with 3 changes))for about 24 hours.

After the fat extraction, the defatted omentum is optionallyre-hydrated. The defatted omentum maybe re-hydrated by contacting thedefatted omentum with a re-hydration solvent, such as alcohol or asolution of alcohol in water, such as an alcohol solution having fromabout 60% to about 70% alcohol. Low molecular weight alcohols, such asmethanol, ethanol, isopropanol, propanol and combinations thereof may beused.

The defatted omentum is then decellularized. Any decellularizationprocess known to one skilled in the art may be applied to decellularizethe defatted omentum. In an embodiment, the defatted omentum may bedecellularized by solubilization of the nuclear and cytoplasmiccomponents. For example, the defatted omentum may be immersed in adecellularization buffer, such as one having non-ionic detergent andmetal salt dissolved in acid for a period of time, typically at leastabout 30 minutes. Non-ionic detergents useful in the invention includepolysorbates, such as TWEEN 80, ethoxylated alcohols, such as TRITON®X-100, and polyethanols, such as HP 40 and IGEPAL CA-630 andcombinations thereof. Metal salts that may be used include magnesiumchloride, phosphate, acetate and citrate, and combinations thereof andthese metal salts are typically dissolved in Tris-HCL.

According to another embodiment, the defatted omentum may bedecellularized by enzymatic proteolytic digestion which digests cellularcomponents within the tissue yet preserves the ECM components (e.g.,collagen and elastin) and thus results in a matrix which exhibits themechanical and structural properties of the original tissue ECM. It willbe appreciated that measures should be taken to preserve the ECMcomponents while digesting the cellular components of the tissue. Thesemeasures are further described herein below and include, for example,adjusting the concentration of the active ingredient (e.g., trypsin)within the digestion solution as well as the incubation time.

Proteolytic digestion according to this aspect of the present inventioncan be effected using a variety of proteolytic enzymes. Non-limitingexamples of suitable proteolytic enzymes include trypsin and pancreatinwhich are available from various sources such as from Sigma (St Louis,Mo., USA). According to one preferred embodiment of this aspect of thepresent invention, proteolytic digestion is effected using trypsin.

Digestion with trypsin is preferably effected at a trypsin concentrationranging from 0.01-0.25% (w/v), more preferably, 0.02-0.2% (w/v), morepreferably, 0.05-0.1 (w/v), even more preferably, a trypsinconcentration of about 0.05% (w/v). For example, a trypsin solutioncontaining 0.05% trypsin (w/v; Sigma), 0.02% EDTA (w/v) and antibiotics(Penicillin/Streptomycin, 1000 units/ml and 0.1 mg/mL respectively),pH=7.2] may be used to efficiently digest all cellular components of thetissue.

Preferably, while in the digestion solution, the tissue segments areslowly agitated (e.g., at about 150 rpm) to enable complete penetrationof the digestion solution to all cells of the tissue.

It should be noted that the concentration of the digestion solution andthe incubation time therein depend on the size of tissue segmentsutilized and those of skilled in the art are capable of adjusting theconditions according to the desired size and type of tis sue.

Preferably, the tissue segments are digested for at least 1 hour and maybe effected for up to 24 hours.

Following decellularization, the omentum may optionally be defattedagain (e.g. using a combination of polar and non-polar solvents).

The method according to this aspect of the present invention optionallyand preferably includes an additional step of removing nucleic acids (aswell as residual nucleic acids) from the tissue to thereby obtain anucleic acid-free tissue. As used herein the phrase “nucleic acid-freetissue” refers to a tissue being more than 99% free of any nucleic acidor fragments thereof as determined using conventional methods (e.g.,spectrophotometry, electrophoresis). Such a step utilizes a DNasesolution (and optionally also an RNase solution). Suitable nucleasesinclude DNase and/or RNase [Sigma, Bet Haemek Israel, 20 μg/ml in Hankbalance salt solution (HBSS)] or combinations of both—e.g. benzonase.High concentration of salts from 0.5M to 3M, such as sodium chloride,can be used also for nucleic acid elimination.

Next, the cellular components are typically removed from the tissue.Removal of the digested components from the tissue can be effected usingvarious wash solutions, such as detergent solutions (e.g., ionic and nonionic detergents such as SDS Triton X-100, Tween-20, Tween-80) which canbe obtained from e.g., Sigma (St Louis, Mo., USA) or Biolab (Atarot,Israel, Merck Germany).

Preferably, the detergent solution used by the method according to thisaspect of the present invention includes TRITON-X-100 (available fromMerck). For efficient removal of all digested cellular components,TRITON-X-100 is provided at a concentration range of 0.05-2.5% (v/v),more preferably, at 0.05-2% (v/v), more preferably at 0.1-2% (v/v), evenmore preferably at a concentration of 1% (v/v).

Optionally, the detergent solution includes also ammonium hydroxide,which together with the TRITON-X-100, assists in breaking and dissolvingcell nuclei, skeletal proteins, and membranes.

Preferably, ammonium hydroxide is provided at a concentration of0.05-1.5% (v/v), more preferably, at a concentration of 0.05-1% (v/v),even more preferably, at a concentration of 0.1-1% (v/v) (e.g., 0.1%).

The concentrations of TRITON-X-100 and ammonium hydroxide in thedetergent solution may vary, depending on the type and size of tissuebeing treated and those of skills in the art are capable of adjustingsuch concentration according to the tissue used. Incubation of thetissue (or tissue segments) with the detergent solution can last from afew minutes to hours to even several days, depending on the type andsize of tissue and the concentration of the detergent solution used andthose of skills in the art are capable of adjusting such incubationperiods. Preferably, incubation with the detergent solution is effectedfor at least 1 hour. According to one embodiment, 1-4 cycles ofincubation with the detergent solution are performed until no foam isobserved.

The above described detergent solution is preferably removed bysubjecting the matrix to several washes in water or saline (e.g., atleast 3 washes), until there is no evidence of detergent solution in thematrix.

Optionally, the decellularized ECM is then sterilized. Sterilization ofthe decellularized ECM may be effected using methods known in the art.In an embodiment, the decellularized omentum is contacted with adisinfection solution for a sufficiently effective period of time todisinfect the decellularized omentum, such as at least about 0.5 hour,typically about 1 hour to about 12 hours. The decellularized omentum maybe fully submerged in the disinfection solution. The disinfectionsolution may comprise alcohol, or an alcohol in water solution, and mayalso include acid. The disinfection solution may include one or more ofthe following ethanol, methanol, isopropanol, propanol, hydrogenperoxide, peracetic acid and combinations thereof. In an embodiment, thedisinfection solution has ethanol, such as 70% ethanol solution.Optionally, the decellularized omentum can be washed one or more timeswith ultrapure water.

Following washing and optional sterilization, the decellularized tissuemay then be dehydrated for example by lyophilization.

Other methods contemplated by the present inventors for decellularizingtissue include those described in U.S. Pat. Nos. 4,776,853, 4,801,299and U.S. Patent Publication No. 20090163990, the contents of each beingincorporated herein by reference in their entirety.

The decellularized omentum of this aspect of the present inventiontypically comprises less than 20% of the cells as compared to the amountof cells in the omentum prior to decellularization, more preferably lessthan 15% of the cells as compared to the amount of cells in the omentumprior to decellularization, more preferably less than 10% of the cellsas compared to the amount of cells in the omentum prior todecellularization, more preferably less than 5% of the cells as comparedto the amount of cells in the omentum prior to decellularization, morepreferably less than 2% of the cells as compared to the amount of cellsin the omentum prior to decellularization.

In one embodiment, the decellularized omentum is devoid of cellularcomponents.

The phrase “devoid of cellular components” as used herein refers tobeing more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, (e.g.,100%) devoid of the cellular components present in the natural (e.g.,native) omentum.

As used herein, the phrase “cellular components” refers to cell membranecomponents or intracellular components which make up the cell. Examplesof cell components include cell structures (e.g., organelles) ormolecules comprised in same. Examples of such include, but are notlimited to, cell nuclei, nucleic acids, residual nucleic acids (e.g.,fragmented nucleic acid sequences), cell membranes and/or residual cellmembranes (e.g., fragmented membranes) which are present in cells of thetissue. It will be appreciated that due to the removal of all cellularcomponents from the tissue, such a decellularized matrix cannot inducean immunological response when implanted in a subject.

The decellularized omentum of this aspect of the present invention isessentially devoid of lipids. The present inventors have found that theextent of extraction of lipids from the tissue correlates with theability to induce cell attachment, maintain cell viability and promoteproper assembly of cells into tissues.

The phrase “devoid of lipids” as used herein refers to a compositioncomprising less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of thelipids present in the natural (e.g., native) omentum.

Solubilization of the decellularized ECM may be effected as described inFreytes et al., Biomaterials 29 (2008) 1630-1637 and U.S. PatentApplication No. 20120156250, the contents of which are incorporatedherein by reference.

Typically, in order to carry out solubilization of the decellularizedomentum it is first dehydrated e.g. lyophilized.

The lyophilized, decellularized omentum may be cut into small pieces,e.g. crumbled, or milled into a powder and then subjected to a secondround of proteolytic digestion. The digestion is effected underconditions that allow the proteolytic enzyme to digest and solubilizethe ECM. Thus, according to one embodiment, the digestion is carried outin the presence of an acid (e.g. HCL) so as to obtain a pH of about 1-4.

Proteolytic digestion according to this aspect of the present inventioncan be effected using a variety of proteolytic enzymes. Non-limitingexamples of suitable proteolytic enzymes include trypsin, pepsin,collagenase and pancreatin which are available from various sources suchas from Sigma (St Louis, Mo., USA) and combinations thereof. Matrixdegrading enzymes such as matrix metalloproteinases are alsocontemplated.

It should be noted that the concentration of the digestion solution andthe incubation time therein depend on the type of tissue being treatedand the size of tissue segments utilized and those of skilled in the artare capable of adjusting the conditions according to the desired sizeand type of tissue.

Preferably, the tissue segments are incubated for at least about 20hours, more preferably, at least about 24 hours. Preferably, thedigestion solution is replaced at least once such that the overallincubation time in the digestion solution is at least 40-48 hours.

Once the decellularized ECM is solubilized, the pH of the solution isincreased so as to irreversibly inactivate the proteolytic enzyme (e.g.to about pH 7). The decellularized, solubilized omentum may be stored atthis stage at temperatures lower than 20° C.—for example 4° C. so thatthe decellularized ECM remains in solution.

The solubilized, decellularized omentum is capable of forming a gel at atemperature above about 30° C., above about 31° C., above about 32° C.,above about 33° C., above about 34° C., above about 35° C., above about36° C., above about 37° C.

The liquid form of the solubilized, decellularized omentum is thenemulsified in oil (water in oil emulsion-W/O) to produce droplets.

In one embodiment, the oil is a fluorinated oil such as a perfluorinatedcarbon oil such as FC-40, FC-770, FC-70, FC-77, FC-72, FC-43, FC-3283,FC-3284, perfluoro-hexane (PFH), perfluoro-cyclohexane (PFC),perfluoro-decaline (PFD), perfluoro-perhydrophenanthrene (PFPH) andNovec/hydrofluoroether (HFE)-7500/7100/7200/71DA/71DE/71IPA/72DA/72DE.

In another embodiment, the oil is a hydrocarbon oil (e.g. light mineraloil, heavy mineral oil, hexadecane, tetradecane, octadecane, dodecane,Isopar™ Isoparaffinic fluids or vegetable oil).

The dispersed phase may be broken into droplets by any method known inthe art including mixing, colloid milling or homogenizing. Surfactantsmay be added (during or following the dispersing phase) in order toimprove the stability of these systems by separating the droplets andmaintaining their shape. Examples of surfactants include, for examplepico-surf™1, pico-surf™2, span80, monoolein, oleic acid, tween 20/80,synperonic, PEF, C12E8, SDS, n-butanol, ABIL EM90 and phospholipids.

The addition of surfactant reduces the surface tension between oil andwater. Therefore, increasing the concentration of surfactant results insmaller oil droplets.

The next step is separation of omentum-gel droplets from the oil phase.In order to do so, the solution may be centrifuged at an appropriatespeed for a number of times so as to remove any residual oil solution.

Another possible way to produce the particles using water in oilstrategy is microfluidic fabrication. This approach allows dropletdimensions to be controlled in a very precise manner by adjusting geland oil phases velocities and by an accurate designing of gel and oilmicrofluidics channels for the most optimal and homogenous dropletcreation. Microfluidics offer many advantages including smallrequirements for solvents, reagents and cells, low cost and versatilityin design.

An additional way to encapsulate cells/biomolecules is using 3Dprinting. Inkjet bio-printing is a “noncontact” technique that useselectromagnetic technology to deposit tiny droplets of “ink” onto asubstrate. Droplet size can be varied by adjusting pulse frequency andink viscosity. Major advantages of 3D printing are high reproducibilityand precise control of droplet size and dose.

Following generation of the particles, the particle is heated so that ithardens. Preferably the particle is heated to a temperature between30-40° C., for example 34-39° C., for example about 37° C.

The solid particles are then removed from the oil by rinsing them usinga hydrophilic solution. In one embodiment, the particles are pipettedwith aqueous/hydrophilic solution and then centrifuged, and. The oilsolution may then be discarded in order to suspend the particles againin an aqueous/hydrophilic solution. The particles can be transferred toaqueous solution with or without addition of perfluoro-1-octanol (PFO,Sigma-aldrich, Rehovot, Israel).

In one embodiment, the particle is then crosslinked.

Chemical crosslinkers such as Carbodiimides (EDC and DCC),N-Hydroxysuccinimide Esters (NHS Esters), Imidoesters, Maleimides,Haloacetyls, Pyridyl Disulfides, Hydrazides, Alkoxyamines, Aryl Azides,Diazirines, Staudinger Reagent Pairs may be used for crosslinking.

Alternatively, or additionally, enzymes such as transglutaminase,sortase, laccase/peroxidase, lysyl oxidase/amine oxidase may be used.Other enzymes are disclosed in Heck et al., Appl Microbiol Biotechnol.2013 January; 97(2): 461-475, the contents of which are incorporated byreference.

Chemical crosslinkers that may be used for the present invention includeCarbodiimides (EDC and DCC), N-Hydroxysuccinimide Esters (NHS Esters),Imidoesters, Maleimides, Haloacetyls, Pyridyl Disulfides, Hydrazides,Alkoxyamines, Aryl Azides, Diazirines, Staudinger Reagent.

According to another embodiment, the particles are sonicated eitherprior to or following the cros slinking step.

The generated particles are typically distinct spheres being of ahomogeneous size. They form a regular shape such that they are capableof being injected without sticking to one another in a syringe.

Thus, according to another aspect of the present invention there isprovided a spherical particle comprising decellularized omentum beingbetween 1 nm-300 μM in diameter.

In one embodiment, the particle is a microparticle (e.g. between 1-300μm, 30-300 μM, 40-300 μM, 50-300 μM or 100-300 μM).

In another embodiment, the particle is a nanoparticle (e.g. 1-1000 nm,10-1000 nm or 100-1000 nm).

According to particular embodiments, more than 10%, 20%, 30%, 40%, 50%of the particle is composed of decellularized omentum.

Therapeutic compounds or agents that modify cellular activity can alsobe incorporated (e.g. encapsulated in, attached to, coated on, embeddedor impregnated) into the particles.

For therapeutic agent incorporation, the agents are added to thesolubilized decellularized ECM of the omentum.

Exemplary agents that may be comprised into the particles of the presentinvention include, but are not limited to those that promote celladhesion (e.g. fibronectin, integrins), cell colonization, cellproliferation, cell differentiation, cell extravasation and/or cellmigration. Thus, for example, the agent may be an amino acid, a smallmolecule chemical, a peptide, a polypeptide, a protein, a DNA, a RNA, alipid and/or a proteoglycan. Proteins that may be incorporated into theparticles of the present invention include, but are not limited toextracellular matrix proteins, cell adhesion proteins, growth factors,cytokines, hormones, proteases and protease substrates. Thus, exemplaryproteins include vascular endothelial-derived growth factor (VEGF),activin-A, retinoic acid, epidermal growth factor, bone morphogeneticprotein, TGFβ, hepatocyte growth factor, platelet-derived growth factor,TGFα, IGF-I and II, hematopoietic growth factors, heparin binding growthfactor, peptide growth factors, erythropoietin, interleukins, tumornecrosis factors, interferons, colony stimulating factors, basic andacidic fibroblast growth factors, nerve growth factor (NGF) or musclemorphogenic factor (MMP). The particular growth factor employed shouldbe appropriate to the desired cell activity. The regulatory effects of alarge family of growth factors are well known to those skilled in theart.

As well as, or instead of, the therapeutic compounds of agents describedherein above, the present invention further contemplates incorporating(e.g. encapsulating) cells into the particles described herein.

For cell incorporation, the cells are added to the solubilizeddecellularized ECM of the omentum.

The cells may be derived from any organism including for examplemammalian cells, (e.g. human), plant cells, algae cells, fungal cells(e.g. yeast cells), prokaryotic cells (e.g. bacterial cells).

Exemplary cells include cardiac cells, neuronal cells, pancreatic cells,stem cells, liver cells, and muscle cells.

According to a particular embodiment the cells comprise stem cells—e.g.adult stem cells such as mesenchymal stem cells or pluripotent stemcells such as embryonic stem cells or induced pluripotent stem cells.The stem cells may be modified so as to undergo ex vivo differentiation.

According to another embodiment, the cells have been differentiated exvivo from induced pluripotent stem cells which have themselves beengenerated from omentum cells.

According to a particular embodiment, the cells are preferably intact(i.e. whole), and preferably viable, although it will be appreciatedthat pre-treatment of cells, such as generation of cell extracts ornon-intact cells are also contemplated by the present invention.

The cells may be fresh, frozen or preserved in any other way known inthe art (e.g. cryopreserved).

In one embodiment, the cells are cardiac cells (e.g. humancardiomyocytes). As used herein, the term “cardiomyocytes” refers tofully or at least partially differentiated cardiomyocytes. Thus,cardiomyocytes may be derived from cardiac tissue or from stem cells(such as embryonic stem cells or adult stem cells, such as mesenchymalstem cells). Methods of differentiating stem cells along a cardiaclineage are well known in the art—[Muller-Ehmsen J, et al., Circulation.2002; 105:1720-6; Zhang M, et al., J Mol Cell Cardiol. 2001; 33:907-21,Xu et al., Circ Res. 2002; 91:501-508, and U.S. Pat. Appl. No.20050037489, the contents of which are incorporated by referenceherein]. According to one embodiment, the stem cells are derived fromhuman stem cell lines, such as H9.2 (Amit, M. et al., 2000. Dev Biol.227:271).

According to one embodiment, the cardiomyocytes of the present inventionare at least capable of spontaneous contraction. According to anotherembodiment, the cardiomyocytes of the present invention express at leastone marker (more preferably at least two markers and even morepreferably at least three markers) of early-immature cardiomyocytes(e.g. atrial natriuretic factor (ANF), Nkx2.5, MEF2C and α-skeletalactin). According to another embodiment, the cardiomyocytes of thepresent invention express at least one marker (more preferably at leasttwo markers and even more preferably at least three markers) of fullydifferentiated cardiomyocytes (e.g. MLC-2V, α-MHC, α-cardiac actin andTroponin I).

Screening of partially differentiated cardiomyocytes may be performed bya method enabling detection of at least one characteristic associatedwith a cardiac phenotype, as described herein below, for example viadetection of cardiac specific mechanical contraction, detection ofcardiac specific structures, detection of cardiac specific proteins,detection of cardiac specific RNAs, detection of cardiac specificelectrical activity, and detection of cardiac specific changes in theintracellular concentration of a physiological ion.

In any of the compositions described herein, the decellularized omentummay be derived from the patient himself (i.e. autologous to the patient)or derived from a subject other than the patient (i.e. non-autologous)and/or the cell populations which are administered to the patienttogether with the decellularized omentum are derived from the patienthimself (i.e. autologous to the patient) or derived from a subject otherthan the patient (i.e. non-autologous).

The particles of the present invention may be used for treating anydisorder associated with tissue degeneration. According to a specificembodiment, the compositions are used for treating a cardiac disorderwhich is associated with a defective or absent myocardium.

Thus, according to another aspect of the present invention there isprovided a method of treating cardiac disorder associated with adefective or absent myocardium in a subject, the method comprisingtransplanting a therapeutically effective amount of the particles of thepresent invention into the subject, thereby treating the cardiacdisorder.

Preferably, the particles of this aspect of the present inventioncomprise (e.g. encapsulate) cardiac cells. The method may be applied torepair cardiac tissue in a human subject having a cardiac disorder so asto thereby treat the disorder. The method can also be applied to repaircardiac tissue susceptible to be associated with future onset ordevelopment of a cardiac disorder so as to thereby inhibit such onset ordevelopment.

The present invention can be advantageously used to treat disordersassociated with, for example, necrotic, apoptotic, damaged,dysfunctional or morphologically abnormal myocardium. Such disordersinclude, but are not limited to, ischemic heart disease, cardiacinfarction, rheumatic heart disease, endocarditis, autoimmune cardiacdisease, valvular heart disease, congenital heart disorders, cardiacrhythm disorders, impaired myocardial conductivity and cardiacinsufficiency. Since the majority of cardiac diseases involve necrotic,apoptotic, damaged, dysfunctional or morphologically abnormalmyocardium, and since the vascularized cardiac tissue of the presentinvention displays a highly differentiated, highly functional, andproliferating cardiomyocytic phenotype, the method of repairing cardiactissue of the present invention can be used to treat the majority ofinstances of cardiac disorders.

According to one embodiment, the method according to this aspect of thepresent invention can be advantageously used to efficiently reverse,inhibit or prevent cardiac damage caused by ischemia resulting frommyocardial infarction.

According to another embodiment, the method according to this aspect ofthe present invention can be used to treat cardiac disorderscharacterized by abnormal cardiac rhythm, such as, for example, cardiacarrhythmia.

As used herein the phrase “cardiac arrhythmia” refers to any variationfrom the normal rhythm of the heart beat, including, but not limited to,sinus arrhythmia, premature heat, heart block, atrial fibrillation,atrial flutter, pulsus alternans and paroxysmal tachycardia.

According to another embodiment, the method according to this aspect ofthe present invention can be used to treat impaired cardiac functionresulting from tissue loss or dysfunction that occur at critical sitesin the electrical conduction system of the heart, that may lead toinefficient rhythm initiation or impulse conduction resulting inabnormalities in heart rate.

The method according to this aspect of the present invention is effectedby transplanting a therapeutically effective amount of the particles ofthe present invention to the heart of the subject (either together withthe cardiac cells or without the cardiac cells).

As used herein, “transplanting” refers to providing the particles of thepresent invention, using any suitable route.

As used herein, a therapeutically effective dose is an amount sufficientto effect a beneficial or desired clinical result, which dose could beadministered in one or more administrations. According to oneembodiment, a single administration is employed. The injection can beadministered into any site in which tissue regeneration is required. Forexample, for treatment of cardiac disorders, the particles can beadministered into various regions of the heart, depending on the type ofcardiac tissue repair required. Intramyocardial administration isparticularly advantageous for repairing cardiac tissue in a subjecthaving a cardiac disorder characterized by cardiac arrhythmia, impaired,cardiac conducting tissue or myocardial ischemia.

Such transplantation directly into cardiac tissue ensures that theadministered cells/tissues will not be lost due to the contractingmovements of the heart.

The particles of the present invention can be transplanted viatransendocardial or transepicardial injection, depending on the type ofcardiac tissue repair being effected, and the physiological context inwhich the cardiac repair is effected. This allows the administered cellsor tissues to penetrate the protective layers surrounding the membraneof the myocardium.

Preferably, a catheter-based approach is used to deliver atransendocardial injection. The use of a catheter precludes moreinvasive methods of delivery wherein the opening of the chest cavitywould be necessitated.

The particles of the present invention can be utilized to regulate thecontraction rate of a heart in response to physiological or metabolicstate of the recipient individual, thereby serving as a biologicalpacemaker.

In the case of repairing cardiac tissue in a subject having a cardiacdisorder characterized by cardiac arrhythmia, electrophysiologicalmapping of the heart and/or inactivation of cardiac tissue byradiofrequency treatment may be advantageously performed in combinationwith administration of the cells and tissues of the present invention ifneeded.

To repair cardiac tissue damaged by ischemia, for example due to acardiac infarct, the particles of the present invention is preferablyadministered to the border area of the infarct. As one skilled in theart would be aware, the infarcted area is grossly visible, allowing suchspecific localization of application of therapeutic cells to bepossible. The precise determination of an effective dose in thisparticular case may depend, for example, on the size of an infarct, andthe time elapsed following onset of myocardial ischemia.

According to one embodiment, transplantation of the compositions of thepresent invention for repair of damaged myocardium is effected followingsufficient reduction of inflammation of affected cardiac tissues andprior to formation of excessive scar tissue.

The present invention can be used to generate cardiomyocytic cells andtissues displaying a desired proliferative capacity, thus cells andtissues are preferably selected displaying a suitable proliferativecapacity for administration, depending on the type of cardiac tissuerepair being effected. Administration of highly proliferative cells maybe particularly advantageous for reversing myocardial damage resultingfrom ischemia since, as previously described, it is the essentialinability of normal adult cardiomyocytes to proliferate which causes theirreversibility of ischemia induced myocardial damage.

Since porcine models are widely considered to be excellent models forhuman therapeutic protocols and since such models have been widelyemployed and characterized, it is well within the grasp of theordinarily skilled artisan to determine a therapeutically effective dosefor a human based on the guidance provided herein, and on that providedby the extensive literature of the art.

Determination of an effective dose is typically effected based onfactors individual to each subject, including, for example, weight, age,physiological status, medical history, and parameters related to thecardiac disorder, such as, for example, infarct size and elapsed timefollowing onset of ischemia. One skilled in the art, specifically acardiologist, would be able to determine the amount and number of cellscomprised in the composition of the present invention that wouldconstitute an effective dose, and the optimal mode of administrationthereof without undue experimentation.

It will be recognized by the skilled practitioner that whenadministering non-syngeneic cells or tissues to a subject, there isroutinely immune rejection of such cells or tissues by the subject.Thus, the method of the present invention may also comprise treating thesubject with an immunosuppressive regimen, preferably prior to suchadministration, so as to inhibit such rejection. Immunosuppressiveprotocols for inhibiting allogeneic graft rejection, for example viaadministration of cyclosporin A, immunosuppressive antibodies, and thelike are widespread and standard practice in the clinic.

In any of the methods described herein, the particles can beadministered either per se or, as a part of a pharmaceutical compositionthat further comprises a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the chemical conjugates described herein, with otherchemical components such as pharmaceutically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of the particles to a subject.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

According to a preferred embodiment of the present invention, thepharmaceutical carrier is an aqueous solution of saline.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

One may administer the pharmaceutical composition in a systemic manner(as detailed hereinabove). Alternatively, one may administer thepharmaceutical composition locally, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art. Depending on the medicalcondition, the subject may be administered with additional chemicaldrugs (e.g., immunomodulatory, chemotherapy etc.) or cells.

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE), etanercept, TNF. alpha. blockers, a biologicalagent that targets an inflammatory cytokine, and Non-SteroidalAnti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are notlimited to acetyl salicylic acid, choline magnesium salicylate,diflunisal, magnesium salicylate, salsalate, sodium salicylate,diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin,ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone,phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen,Cox-2 inhibitors and tramadol.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods

Device design: Microfluidic devices were fabricated by soft lithography.Negative photo resist SU-8 (3050, MicroChem, Corp. Newton, Mass.) wasspin-coated onto a clean silicon wafer (300 μm thick, University Wafer,Boston, Mass.) to a thickness of 50 μm and patterned by UV exposurethrough a transparency photomask. After developing the microstructure, adegassed 10:1 mixture of Sylgard 184 poly(dimethylsiloxane) (PDMS)(Sylgard 184, Dow Corning Corp. Midland, Mich.) and cross-linker (ratio10:1) was poured onto the pattern, degassed and cured for 1 hour at 65°C. The PDMS molds were peeled off the master and the channel inlets andoutlets were made by using a 0.75 mm diameter biopsy punch (WorldPrecision Instruments, Sarasota, Fla.). The PDMS replicas were bonded toa glass slide after oxygen-plasma activation of both surfaces usingoxygen plasma (Diener Electronic GmbH & Co. KG, Germany). To avoidwetting of the channels by the dispersed phase, the devices were treatedwith Aquapel (PPG Industries, Pittsburgh, Pa., USA) by flushing thechannels with the solution as received and air dried immediately.

Decellularization of the omentum: Omenta of healthy pigs were purchasedfrom the institute of animal research in Kibutz Lahav, Israel. The freshtissues were washed with phosphate buffered saline (PBS) in order todeplete blood and debris. Then, the omentum was agitated for 1 hour in ahypotonic buffer of 10 mM Tris 5 mM Ethylenediaminete-traacetic acid(EDTA) and 1 μM phenylmethanesulfonyl-fluoride (PMSF) at pH 8.0. Next,the tissue went through three cycles of freezing (−80° C.) and thawing(37° C.) using the same buffer. After the last thawing, the tissue wasdehydrated by washing it once with 70% ethanol for 30 minutes and threetimes in 100% ethanol for 30 minutes each. Then the polar lipids of thetissue were extracted by three 30 min washes of 100% acetone. Finely theapolar lipids were extracted by 24 hour incubation in a 60/40 (v/v)hexane:acetone solution (with 3 changes). The defatted tissue wasrehydrated by one 30 minute wash in 100% ethanol and an overnightincubation in 70% ethanol at 4° C. Then the tissue was washed four timeswith PBS at pH 7.4 and was incubated in 0.25% Trypsin-EDTA (BiologicalIndustries, Kibbutz Beit-Haemek, Israel) solution overnight. The tissuewas then washed thoroughly with PBS and incubated with 1.5 M NaCl for 24hours (3 changes) for nucleic acid degradation. Finally the tissue waswashed with 50 mM Tris 1% triton-X100 solution at pH 8.0 for 1 hour. Thedecellularized tissue was washed three times with PBS and three timeswith double distilled water. The decellularized tissue was frozen (−20°C.) and lyophilized.

Preparation of solubilized omentum dECM: After lyophilization, thedecellularized omentum was ground into a coarse powder using a WileyMini-Mill and then frozen until further use. Dry, milled omentum dECMwas enzymatically digested by adding a 1 mg ml⁻¹ solution of pepsin(Sigma, 3200-4500 units mg⁻¹ protein) in 0.1 M HCl. The finalconcentration of dECM was 1% (w/v). The dECM was digested for 96 h at RTunder constant stirring until the liquid was homogenous with no visibleparticles. Subsequently, the pH was raised to 6.5 using 5 M NaOH, thenDMEM/F12(HAM) X10 (Biological industries, Beit-Haemek, Israel) was addedand the pH was raised again to 7.2-7.4. Raising the pH terminates pepsinactivity (the enzyme is deactivated above pH 6).

Rheological properties: Rheological experiments were performed using aDiscovery HR-3 hybrid Rheometer (TA Instruments, DE) with 8 mm diameterparallel plate geometry with a Peltier plate to maintain the sampletemperature. The samples were loaded into the rheometer with the Peltierplate maintaining a temperature of 4° C. The sample was protected fromevaporation by wetting of the chamber cover. The temperature was thenset to 37° C. to induce gelation; during this time the oscillatorymoduli of the sample were monitored continuously at a fixed frequency of0.127 Hz and a strain of 1%.

Droplet generation: To fabricate omentum-hydrogel droplets, the liquidomentum-hydrogel mixture was loaded into 1 ml sterile disposable syringe(pic solution, Italy). A mixture of 2% Pico-Surf 2 surfactant inperfluorinated carbon oil (Sphere fluidics, Cambridge, United kingdom)was used as the outer phase. Fine Bore Polythene tubings (Smiths MedicalInternational Ltd., Kent, UK) with an outer diameter of 1.09 mm and aninner diameter of 0.38 mm was used to connect the channel inlets withthe syringes. Flow rates were controlled by NE-1000 programmable singlesyringe pump (New era pump systems, USA).

Droplet generation was monitored with a digital microscope (Dino-litedigital microscope, New Taipei city, Taiwan). The flow rates wereindividually adjusted to obtain the aspired droplet size. Generateddroplets were collected from the outlet channel and transferredimmediately to 37° C. for four minutes, for their gelation. Aftergelation, the droplets were transferred into aqueous medium by simplesuspension of the droplets.

Following a three minute centrifugation at 600 RPM, the oil phase wasdiscarded and the droplets were re-suspended in culture medium.

Cell culture-3T3 fibroblasts: 3T3 fibroblasts cells were grown inDulbecco' s Modified Eagle Medium (DMEM) supplemented with 10% (v/v)fetal bovine serum (FBS, Biological Industries, Beit-Haemek, Israel) and1% (v/v) Penicillin/Streptomycin. The cells were split every five daysunder sterile conditions and incubated at 37° C. and 5% CO₂.

Cardiomyocyte isolation: The left ventricles of 0-3 d-old neonatalSprague-Dawley rats were harvested and the cells were isolated using 6cycles (30 min each) of enzyme digestion with collagenase type II (345U/mg dW, Worthington biochemical corporation, NJ, USA) and pancreatinfrom porcine pancreas (Sigma-Aldrich, Rehovot, Israel) in DMEM. Aftereach round of digestion, cells were centrifuged (600 g, 5 min) andre-suspended in culture medium composed of M-199 (Biological Industries,Beit-Haemek, Israel) supplemented with 0.6 mM CuSO₄.5H₂O, 0.5 mMZnSO₄.7H₂O, 1.5 mM vitamin B12, 500 U mL⁻¹ penicillin and 100 mg mL⁻¹streptomycin, and 0.5% (v/v) fetal bovine serum (FBS). To enrich thecardiomyocyte population, cells were suspended in culture medium with 5%FBS and pre-plated twice for 50 minutes.

Spinal cord motor neurons: Spinal cord motor neurons were differentiatedfrom omentum-derived hiPSCs.

Encapsulation: Prior to encapsulation, cells were counted, centrifugedand resuspended in culture medium to gain the desired cell density andsuspended in 9:1 omentum-hydrogel:culture medium mix. The hydrogelmixture was then loaded into a plastic 1 ml syringe for dropletgeneration as described above. Following gelation, the microgels weretransferred into aqueous followed by centrifugation (600 rpm, 3minutes). The resulting oil phase was discarded and the droplets werere-suspended in cell culture medium. Cell-laden droplets were incubatedat 37° C. under 5% CO₂. FDA/PI was used to determine the cell viability.

Scanning electron microscopy: For scanning electron microscope (SEM)imaging, samples of cell-laden droplets were fixed with 2.5%glutaraldehyde in PBS for 24 hours at 4° C., followed by dehydrationusing a graded series of ethanol-water solutions (50-100%). All sampleswere critical point dried, sputter-coated with gold in a Polaron E 5100coating apparatus (Quorum technologies, Laughton, UK) and observed underJSM-840A SEM (JEOL, Tokyo, Japan).

Results

Device design: Flow-focusing microfluidics device was designed toencapsulate omentum-gel droplets ca. 50-300 μm in diameter with orwithout cells. The device has two inlets of surfactant/oil combination(continuous phase) and omentum-hydrogel (dispersed phase), respectively,and one outlet in order to collect the produced droplets (FIG. 1).

Bright Field (BF) microscopy images of the device were taken during theencapsulation process of 3T3 fibroblasts (a model cell line). As can beseen in FIG. 2A, droplets were pinched-off, by the two oil streams, inthe droplet production zone. Droplets were stabilized by the surfactantwhile flowing to the outlet stream of the device (FIG. 2B).

Omentum-hydrogel rheological measurements: Rheological properties of theomentum-hydrogel is important characteristics in order to predict itsbehavior under applied forces. When the temperature was elevated from 4to 37° C. and during the gelation period, both the storage modulus (G′)and the loss modulus (G″) increased over time, and were characterized bya sigmoidal shape (FIG. 3). As G′ was greater than G″ throughout themeasurements, the omentum-hydrogel possess pronounced elastic gelproperties, which is essential for cardiac engineered tissue.

Optimization of pico-surf™2 surfactant in FC-40 oil for maintaining cellviability: Different percentages of pico-surf™ 2 surfactant were tested.The viability of encapsulated 3T3 fibroblasts cells was investigated inorder to prove that the encapsulation process is not toxic to the cells.As can be seen in FIG. 4A, high viability percentages were achieved forboth surfactant concentrations. In addition, droplet diameter wasmeasured for each experiment to examine the size distribution of theencapsulation process. As can be seen in FIGS. 4B and 4C, dropletdiameter mean size, using 5% and 2% pico-surf™2, was 73.71 μm and 67.86μm, respectively. The droplet mean size was reproducible using 5% and 2%surfactant and Gaussian size distribution was achieved for both. Using5% surfactant, a wider distribution can be seen (FIG. 4B), which may beattributed to chunks of omentum-hydrogel plugging the channel of thedispersed phase, leading to changes in the dispersed phase velocitywhich resulting in disrupted flow profile in the nozzle zone. Sincethere were no significant differences between the 5% and 2% surfactant,2% pico-surf™2 was chosen for further experiments.

Dispersed phase velocity effect on droplet production and cellviability: Through changing continuous and dispersed phase velocities,the droplet size can be controlled. Thus, dispersed phase velocitieswere changed and cell viability and droplet diameter were examined. Ascan be seen in FIG. 5A, viability of encapsulated 3T3 fibroblasts cellswas excellent for each of the velocities. Mean droplet diameter using20, 40 and 60 μl hr⁻¹ dispersed phase velocity, were 64.01, 67.86 and79.42 μm, respectively (FIG. 5B). As expected, as the dispersed phasevelocity was increased, the droplet diameter increased as well. Using avelocity of 20 μl hr⁻¹ (FIG. 5C), a narrow Gaussian distribution wasachieved. Higher velocities (40 and 60 μl hr ⁻¹, FIGS. 5D and 5E,respectively) resulted in wider Gaussian distribution, reaching 150 μmsize droplets.

As can be seen in FIG. 6A, significant amount of homogenous droplets wasproduced while a high percentage contained encapsulated cells. Thesurfactant preventing coalescence of the droplets, resulting in stable,well ordered droplets. After separation of droplets into an aqueousmedium, live/dead assay was conducted, for the quantification of cellviability. FIG. 6B is a visual example for the high cell viability thatwas achieved using the present system.

Continuous phase velocity effect on droplet production and cellviability: Continuous phase velocity was changed and cell viability anddroplet diameter were examined. As can be seen in FIG. 7A, viability ofencapsulated 3T3 fibroblasts cells was excellent irrespective of thecontinuous phase velocity. Mean droplet, using 60, 80 and 100 μl hr⁻¹continuous phase velocity, was 77.6, 74.2 and 73.2 μm, respectively(FIG. 7B). There was a good correlation between continuous phasevelocity and droplet diameter. As continuous phase velocity increased,so droplet diameter decreased. Using a velocity of 60 μl hr⁻¹ (FIG. 7C),a wide Gaussian distribution was reached. The oil velocity is too low inorder to pinch the droplets homogenously. Higher velocities (80 and 100μl hr ⁻¹, FIGS. 7D and 7E, respectively) resulted in narrower Gaussiandistribution, reaching 65% of droplets in diameter of 70-75 μm. Theeffect of increasing continuous phase velocity was not dominant asincreasing dispersed phase velocity, as expected. It is attributed tothe higher viscosity of the omentum-hydrogel compared to the oil phaseviscosity.

Cell concentration effect on droplet production and cell viability: Cellconcentration was changed and cell viability and droplet diameter wereexamined. As can be seen in FIG. 8A, viability of encapsulated 3T3fibroblasts cells was not affected by cell concentration. Using 25×10⁶and 50×10⁶cells mL⁻¹, the number of cells per droplet was 4.4 and 17,respectively (FIG. 8B). Using 25×10⁶ and 50×10⁶ cells mL⁻¹, a narrowGaussian distribution was achieved (FIG. 8C and 8D, respectively).

SEM images of omentum-hydrogel droplets: In order to confirm omentumfiber existence and its support to the cells, after droplet productionand separation, omentum-hydrogel droplets with and without encapsulatedcells were imaged using SEM. As can be seen in FIGS. 9A-B, cells wereencapsulated within the omentum-hydrogel in a way that each cell issurrounded by the omentum-fibers, providing mechanical and biochemicalsupport.

Immunofluorescence staining of encapsulated cells in omentum-hydrogeldroplets: hiPSCs were differentiated to spinal cord neurons in cultureuntil day 19, then encapsulated within omentum-hydrogel droplets andwere cultured whilst being differentiated until day 28. On day 30,droplets were fixed and encapsulated neurons were stained for β-tubulinand nuclei while the omentum-hydrogel droplets were stained for collagenI. The encapsulated cells are illustrated in FIGS. 10A-C.

In addition, neonatal cardiomyocytes (CMs) were isolated andencapsulated within omentum-hydrogel droplets, fixed at day 8 (FIGS.11A-B) and day 13 (FIGS. 11C-D) and were stained for collagen I, nucleiand actinin. As can be seen in FIGS. 11A-D, all CMs spread evenly withinthe omentum-hydrogel droplet.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. A spherical particle comprising decellularized omentum being between1 nm-300 μM in diameter.
 2. The particle of claim 1, being amicroparticle.
 3. The particle of claim 2, being 50-300 μm in diameter.4. (canceled)
 5. The particle of claim 1, wherein more than 20% of theparticle is composed of decellularized omentum.
 6. The particle of claim1, wherein said omentum comprises human omentum.
 7. The particle ofclaim 2, encapsulating at least one biological cell.
 8. The particle ofclaim 7, wherein said biological cell is selected from the groupconsisting of a cardiac cell, a neuronal cell, a pancreatic cell, a stemcell, a liver cell, a muscle cell, a blood cell and an immune cell. 9.The particle of claim 1, encapsulating at least one biomolecule.
 10. Theparticle of claim 7, further encapsulating at least one biomolecule. 11.(canceled)
 12. A composition comprising: (i) a plurality of theparticles of claim 1; and (ii) biological cells and/or at least onebiomolecule.
 13. The composition of claim 12, wherein each of theparticles of said plurality of particles are of substantially the samesize.
 14. The composition of claim 12, wherein said particlesencapsulate said biological cells and/or at least one biomolecule.15-16. (canceled)
 17. A method of generating particles comprisingdecellularized omentum comprising: (a) dispersing a compositioncomprising solubilized decellularized omentum in an oil under conditionsthat allow generation of emulsified, decellularized omentum; and (b)heating said emulsified, decellularized omentum to generate solidparticles of decellularized omentum, thereby generating particlescomprising decellularized omentum.
 18. The method of claim 17, furthercomprising separating said solid particles comprising decellularizedomentum from said oil following said heating.
 19. The method of claim17, wherein said heating is effected at about 37° C.
 20. The method ofclaim 17, wherein said oil is a fluorinated oil.
 21. The method of claim20, wherein said fluorinated oil is selected from the group consistingof FC-40, FC-770, FC-70, FC-77, FC-72, FC-43, FC-3283, FC-3284,perfluoro-hexane (PFH), perfluoro-cyclohexane (PFC), perfluoro-decaline(PFD), perfluoro-perhydrophenanthrene (PFPH) and Novec/hydrofluoroether(HFE)-7500/7100/7200/71DA/71DE/71IPA/72DA/72DE.
 22. The method of claim17, wherein said oil is a hydrocarbon oil.
 23. The method of claim 22,wherein said hydrocarbon oil is selected from the group consisting oflight mineral oil, heavy mineral oil, hexadecane, tetradecane,octadecane, dodecane, Isopar™ Isoparaffinic fluids and vegetable oil.24-25. (canceled)
 26. The method of claim 18, wherein said separatingsaid solid particles of decellularized omentum from said oil is effectedby contacting said solid particles with a hydrophilic solution. 27-28.(canceled)
 29. The method of claim 18, further comprising sonicatingsaid solid particles following said separating.
 30. The method of claim17, wherein said composition further comprises at least one biologicalcell.
 31. (canceled)
 32. The method of claim 17, wherein saidcomposition further comprises a biomolecule. 33-37. (canceled)
 38. Themethod of claim 18, further comprising crosslinking said particlesfollowing said separating.
 39. A method of treating a disease or medicalcondition which would benefit from cell transplantation in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of a plurality of the particles of claim 1, therebytreating the medical condition.